The long-term goals of our research are to understand the mechanisms of HPV-host interactions necessary for establishing and maintaining HPV infections in human keratinocytes throughout HPV-associated cancer progression. Human papillomaviruses (HPVs) are the most common sexually transmitted pathogen, causally associated with a growing number of human cancers including cervical, vaginal, penile, anal, head/neck, and other squamous carcinomas, causing almost half a million deaths worldwide each year (International Agency for Research on Cancer). To establish persistent infections, the virus must enter the nucleus and establish its genome as a nuclear plasmid. However, little is known about how HPV genome enters the nucleus and establishes its replication, due to technical limitations. To achieve our goals, we will: 1) define the role of host cell mitosis and microtubule reorganization for HPV DNA nuclear entry in human keratinocytes; and 2) investigate host innate responses and its roles for early steps of HPV infection in primary keratinocytes in vitro and in vivo. In these studies, we will develop reliable HPV DNA labeling systems with various fluorescent tags and track the movement of HPV DNA in live human keratinocytes, using infectious HPV virions and real time imaging technology. In addition, we will test alterations of HPV infectivity, DNA entry into the nucleus, and genome replication by knockdown of host factors involved in mitosis, microtubule network, and host innate immunity. Recently developed prophylactic HPV vaccines should eventually reduce infections by some HPV genotypes. However, because the vaccines do not cover all HPVs and do not eliminate existing infections, there remains an urgent need to develop new means of intervening in HPV infections and preventing cancer progression. Our studies will provide further understanding of mechanisms employed by HPVs to establish infections and thereby identify pathways to target for preventing HPV-associated cancers.